In vitro nucleic acid amplification techniques provide powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids as in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR; R. K. Saiki, et al. 1985. Science 230, 1350-1354), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. U.S.A. 88, 189-193) and transcription-based amplification (D. Y. Kwoh, et al. 1989. Proc. Natl. Acad. Sci. U.S.A. 86, 1173-1177) require temperature cycling. In contrast, methods such as Strand Displacement Amplification (SDA; G. T. Walker, et al. 1992. Proc. Natl. Acad. Sci. U.S.A. 89, 392-396; G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696; U.S. Pat. No. 5,270,184), self-sustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878) and the Q.beta. replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. In addition, WO 90/10064 and WO 91/03573 describe use of the bacteriophage phi29 replication origin for isothermal replication of nucleic acids.
A variety of methods have been developed to detect and/or measure nucleic acid amplification. For the most part, these methods are primer-based, meaning that they depend on hybridization of a primer to the target sequence, in some cases followed by extension of the primer. Primer-based detection of amplified nucleic acids in PCR often relies on incorporation of an amplification primer into the amplified product (amplicon) during the amplification reaction. Features engineered into the PCR amplification primer therefore appear in the amplification product and can be used either to detect the amplified target sequence or to immobilize the amplicon for detection by other means. However, primer-based methods of detecting PCR amplification products require two amplification reactions to achieve high sensitivity, i.e., detection of fewer than 100 copies of the target sequence. That is, a first amplification of the target sequence is followed by a second amplification using nested primers incorporating the desired modifications for capture and/or detection. Two consecutive amplifications are needed to avoid unacceptably high levels of background signal produced by amplification of non-target DNA spuriously primed with the modified, signal-generating primers. This feature of the prior art methods makes them time-consuming and cumbersome, and the advantages of primer-based detection methods are therefore often offset by the requirement for a second consecutive amplification reaction.
P. M. Holland, et al. (1992. Clin. Chem. 38, 462-463) describe a method for detecting amplification products of PCR in which the 5'-3' exonuclease activity of Taq DNA polymerase is used to generate target amplification-specific signal by digestion of a labeled probe hybridized downstream of the amplification primer. The labeled probe is not extendable, possibly because certain of the detection systems described make use of a 3' end-label. Further, an extendable labeled probe would function as a PCR amplification primer, thereby increasing non-specific background signal in the reaction. Cleaved probe fragments are generated during amplification, and may be differentiated from uncleaved probe in a variety of ways, depending on the type of probe label. The authors suggest thin-layer chromatography or capture by a 3' biotin label to separate cleaved from uncleaved probe, or sequencing. These detection methods require cumbersome and time consuming manipulations of the sample after amplification. The present methods for primer-based detection of target amplification also make use of a single amplification reaction to concurrently generate secondary products for detection. In contrast to P. M. Holland, et al. and other prior art methods, however, the secondary amplification products are detected in a simple format by extraction into an organic phase.
As used herein, the following terms and phrases are defined as follows:
An amplification primer is a primer for amplification of a target sequence by primer extension. For SDA, the 3' end of the amplification primer (the target binding sequence) hybridizes at the 3' end of the target sequence. The amplification primer comprises a recognition site for a restriction endonuclease near its 5' end. The recognition site is for a restriction endonuclease which will cleave one strand of a DNA duplex when the recognition site is hemimodified ("nicking"), as described by Walker, et al. (1992. PNAS, supra). A hemimodified recognition site is a double stranded recognition site for a restriction endonuclease in which one strand contains at least one derivatized nucleotide which causes the restriction endonuclease to nick the primer strand rather than cleave both strands of the recognition site. Usually, the primer strand of the hemimodified recognition site does not contain derivatized nucleotides and is nicked by the restriction endonuclease. Alternatively, the primer may contain derivatized nucleotides which cause the unmodified target strand to be protected from cleavage while the modified primer strand is nicked. Such restriction endonucleases can be identified in routine screening systems in which a derivatized dNTP is incorporated into a restriction endonuclease recognition site for the enzyme. The preferred hemimodified recognition sites are hemiphosphorothioated recognition sites for the restriction endonucleases HincII, HindII, AvaI, NciI, Fnu4HI, BsoBI and BsrI. The amplification primer also comprises a 3'--OH group which is extendable by DNA polymerase when the target binding sequence of the amplification primer is hybridized to the target sequence. For the majority of the SDA reaction, the amplification primer is responsible for exponential amplification of the target sequence. As no special sequences or structures are required, amplification primers for PCR generally consist only of target binding sequences.
Extension products are nucleic acids which comprise a primer and a newly synthesized strand which is the complement of the target sequence downstream of the primer binding site. Extension products result from hybridization of a primer to a target sequence and extension of the primer by polymerase using the target sequence as a template.
A bumper primer is a primer which anneals to a target sequence upstream of the amplification primer, such that extension of the bumper primer displaces the downstream amplification primer and its extension product. Extension of bumper primers is one method for displacing the extension products of amplification primers, but heating is also suitable.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, its complementary second strand and either strand of a copy of the original sequence which is produced in the amplification reaction. The target sequence may also be referred to as a template for extension of hybridized primers.
A signal primer is a primer which hybridizes to a target sequence downstream of an amplification primer such that extension of the amplification primer displaces the signal primer, a portion of the signal primer or the signal primer extension product. The signal primer further comprises a lipophilic reporter group or label which facilitates detection of secondary amplification products generated from the signal primer.
Amplification products, amplified products or amplicons are copies of the target sequence generated by hybridization and extension of an amplification primer. This term refers to both single stranded and double stranded amplification primer extension products which contain a copy of the original target sequence, including intermediates of the amplification reaction.
Secondary amplification products or secondary products are oligonucleotides generated from a signal primer in a target amplification-dependent manner. These terms refer to single stranded or double stranded products generated from signal primers, as well as portions of signal primers or signal primer extension products generated as a result of target amplification.
Cleavage of an oligonucleotide refers to breaking the phosphodiester bonds of the molecule such that two oligonucleotide cleavage products are produced, i.e., breaking the bonds of both strands of a DNA duplex or breaking the bond of single-stranded DNA. This is in contrast to nicking, which refers to breaking the phosphodiester bond of only one of the two strands in a DNA duplex.